Mercuric ion is extremely cytotoxic to both procaryotic and eucaryotic cells (Umeda et al., 1969; Umeda and Nishimura, 1979; Cantoni et al., 1982; Goldberg et al., 1983; Cantoni et al., 1984). In humans, mercuric ion is known to accumulate in the thyroid gland and to potentially lead to the formation of pre-malignant and malignant thyroid nodules (Zaichick et al., 1985). The precise mechanism(s) mediating the carcinogenic effects of mercuric ion is not well defined, but appears to be related to the ingestion or inhalation of the metal, conversion of the metal to mercuric ion, and the translocation of the ion to the thyroid (Zaichick et al., 1985). Part of the cytotoxic and carcinogenic effects of mercuric ion may be due to the ability of the metal ion to alter the DNA synthetic machinery of the cell (Goldberg et al., 1983; Robinson et al., 1984; Williams et al., 1986). Alterations of the activity of the DNA replication machinery are suggested to have a role in mediating the mutagenic effects of mercuric ion (Ariza and Williams, 1996).
Mercuric ion has been reported to alter both the extent of DNA synthesis and the type of DNA replication formed in experiments using intact mammalian cells, (Ariza and Williams, 1996; Robinson et al., 1984; Oberly et al., 1982; Christie et al., 1984), crude mammalian cell extracts, (Oberly et al., 1982; Robinson et al., 1984) and purified enzymes (Caldentey et al., 1992; Williams et al., 1986; Oberly et al., 1982; Niyogi et al., 1981; Hsie et al., 1979). In addition, studies using purified bacterial phage DNA polymerase P1 is strongly inhibited by mercuric ion (Cladentey et al., 1992). The inhibition of DNA polymerase activity by mercuric ion is postulated to be at least partially responsible for the observed inhibition of intact cell DNA synthesis. Furthermore, mercuric ion not only inhibits the activity of the DNA polymerase, but alters the fidelity with which DNA synthesis is carried out by this polymerase.
In vitro mutagenesis assays utilizing purified E. coli DNA polymerase have demonstrated that several divalent metals, which are known to be carcinogenic (e.g. lead, cadmium, and nickel) produce significant increases in the number of nucleotide misincorporations during the DNA synthesis process (Sirover and Loeb, 1976; Miyaki et al., 1977; Sirover et al., 1979; Tkeshelashvili et al., 1979; and Tkeshelashvili et al., 1980). Because mercuric ion is also a suspected carcinogen and is a member of the group BII elements, which also contains cadmium ion, we decided to examine whether the mercuric ion could potentially act as a carcinogen by altering the activity of the DNA synthetic machinery of the cell.
Mercuric ion has-been postulated to use one or more of the following mechanisms to alter the fidelity of the DNA synthesis process. First, mercuric ion can alter substrate conformation (i.e. through metal-nucleotide interaction); second, it can alter the conformation of proteins essential for replication and for repair (Williams and Crothers, 1975) (i.e. through metal-protein interactions); third, mercuric ion can alter template-base specificity, (Zakour et al, 1981). Mercuric ion also exhibits two properties which dramatically contribute to the development of alterations contained in cells exposed to mercuric ion. First, mercuric ion has been shown to have potent DNA strand scission activity (Cantoni et al., 1989; Robinson et al., 1984; and Williams et al., 1986). This property allows the ion to induce changes in the DNA template which can alter the ability of the DNA replication machinery to bind to the template properly. This disruption in template binding is also postulated to distort at least some of the components of the replication machinery, and subsequently alter the binding of deoxynucleotides by DNA polymerase and cause misincorporation of nucleotides into the growing DNA strand. Second, mercury has a strong affinity for thiol bonds, which are present in virtually all of the replication proteins, and many other cellular enzymes (Hayes, 1983). The binding of mercuric ion to these thiol groups can severely distort the structural integrity and activity of these proteins. To examine whether mercuric ion can alter the activity and fidelity of the DNA synthetic apparatus of human cells we isolated the cellular DNA synthesizing machinery from human cervical cancer cells (HeLa) and tested the effects of a range of mercuric ion concentrations on in vitro DNA replication activity, DNA polymerase activity, and fidelity with which this complex carries out DNA synthesis.
We chose to use the isolated DNA synthetic machinery from HeLa cells for these studies because our characterization of this highly organized complex of proteins (which we have termed the DNA synthesome) (Lin et al., 1996) indicated that the DNA synthesome contained DNA polymerases .alpha. and .delta.. The synthesome is fully competent to support all phases of simian virus 40 (SV40) origin-specific DNA replication in vitro (Malkas et al., 1990). The biochemical characterization of this isolated multiprotein form of DNA polymerase has resulted in the identification of several protein components of the synthesome. These proteins include: DNA polymerases .alpha., .delta., .epsilon., DNA primase, topoisomerases I and II, proliferating cell nuclear antigen (PCNA), replication factor C (RFC), replication protein A (RPA), DNA helicase, DNA methyltransferase, poly(ADP)ribose polymerase and DNA ligase I (Malkas et al., 1990; Applegren et al., 1995; Coll et al., 1996). In addition, the DNA replication process mediated by the human cell synthesome in vitro has been shown to exhibit all of the features of the replication process as is carried out by intact cell (Malkas et al., 1990; Applegren et al., 1995). The DNA synthesome has been isolated and characterized from human and murine cells (Malkas et al., 1990; Applegren et al., 1995; Wu et al., 1994), human breast tissue cells and primary human breast tumors (Coll et al., 1996), from human leukemia cells (Lin et al., 1996), and from human pancreatic cells (Hickey, unpublished data). Our model describing the DNA synthesome is based on the sedimentation and chromatographic profiles of the individual proteins found to co-purify with one another as a fully functional DNA replication complex (Wu et al., 1994; Applegren et al., 1995, Coll et al., 1996).
The results presented here strongly suggest that the human DNA synthesome can serve as a useful and highly novel in vitro model system for testing whether heavy metal ions can directly induce changes in the activity and fidelity of the cellular DNA synthetic apparatus.